KRAS is one of the most deadly, yet undrugged, cancer proteins and is present in over 30% of all human tumors, with even higher frequencies found in pancreatic, lung, thyroid, colon, and liver cancers. Thus, achieving new mechanistic insights into KRAS deregulation and advancing innovative approaches to neutralize oncogenic KRAS remain among the highest priorities of the cancer field and represent the focus of this interdisciplinary proposal. KRAS is a GTPase that serves as a critical control point for a host of cellular functions ranging from cell survival and proliferation to endocytosis and motility. The functional activity of KRAS is dictated by nucleotide exchange, with the GTP-bound and GDP-bound forms representing the on and off states, respectively. Cancer cells hijack and enforce the activated state of KRAS through gain-of-function mutagenesis or gene amplification. To date, small molecule approaches to directly block the GTP-binding site have been unsuccessful due to subnanomolar engagement of GTP and GDP by KRAS. The structure of KRAS in complex with SOS1, a guanine nucleotide exchange factor that enhances KRAS activity by facilitating GDP release, revealed a helix-in-groove interaction potentially targetable by ?-helical mimicry. We applied all-hydrocarbon peptide stapling to generate stabilized alpha-helices of SOS1 (SAH-SOS1) and identified a prototype compound that engaged oncogenic KRAS, including the broad diversity of clinical mutants, inhibited the ERK-MAP kinase phosphosignaling cascade downstream of KRAS, and impaired the viability of KRAS-driven cancer cells. We found that not only did the prototype SAH-SOS1 construct dissociate the catalytic SOS1/KRAS interaction as anticipated, but also directly and independently blocked nucleotide association with KRAS by an unknown mechanism. Here, we aim to apply chemical, structural, cellular, and in vivo approaches to interrogate just how a SAH-SOS1 peptide can directly block the enzymatic activity of KRAS, compare and contrast this mechanism to the natural agonist activity of the SOS1 protein, and thereby inform both our structure-function understanding of SOS1/KRAS regulation and a new strategy for therapeutic inhibition of KRAS in human cancer. To achieve these goals, we propose three experimental aims: (1) Synthesize an expansive library of structurally-reinforced helices modeled after the KRAS-interaction domain of SOS1 to identify the binding determinants and functional interactions with KRAS and its oncogenic mutants; (2) Apply hydrogen-deuterium exchange mass spectrometry to elucidate the conformational effects of the SOS1 protein and SAH-SOS1 peptides on KRAS proteins and thereby define the mechanisms of enzymatic regulation; (3) Advance optimized SAH-SOS1 inhibitors to cellular and in vivo testing in KRAS-driven cancers to validate mechanism of action and therapeutic window, and provide proof-of-concept for clinical translation. By combining the biochemical and mass spectrometry expertise of the Engen laboratory with the cancer chemical biology and translational approaches of the Walensky laboratory, our goal is to provide new mechanistic insight into the oncogenic KRAS pathway and inform a new modality to disarm it for therapeutic benefit in cancer.